The long term goal of this research project is to understand the translocation mechanisms which transfer the A1 polypeptide of cholera toxin (CTA1) from the endomembrane system to the cytosol of eukaryotic cells. It has been proposed that translocation occurs across the endoplasmic reticulum (ER) membrane and involves the action of ER-associated degradation (ERAD). This quality control mechanism recognizes misfolded proteins in the ER lumen and exports them into the cytosol for ubiquitination and degradation by the proteasome. This project will test the validity of this model. The first part of the project will involved the establishment of a translocation assay. Once established, the assay will be used to identify the determinants of cholera toxin essential for translocation. In addition, an attempt will be made to identify cellular factors required for toxin translocation by isolating cell lines resistant to the toxin and subsequent identification of the mutated genes. It is expected that this project will not only help the understanding of cholera toxin translocation but also the translocation of other related toxins as well.